Specific molecular recognition is a fundamental process, being the basis of enzyme-ligand interactions, antibody-antigen interactions and the binding of molecules to receptors. Molecular recognition is achieved through non-covalent interactions such as electrostatic interaction (hydrogen bonds) and hydrophobic interactions. Thermodynamic measurements of binding constants and free energy, enthalpy and entropy changes offer insight into the molecular basis of recognition, particularly when coupled with information from X-ray diffraction and, when possible, site-directed mutagenesis.
Direct measurement of the force of interaction has been made by atomic force microscopy (AFM) as well as surface force apparatus. While AFM is capable of measuring bond rupture forces, the technique has the disadvantage that only one measurement can be made at a time. To date, AFM has been used on avidin-biotin interactions (Florin et al., Science, 1995; 264:415), DNA hybridisation (Boland et al, PNAS, 1995; 92:5291), antibody-antigen interactions (Dammer et al, Biophys. J., 1996; 70:2437) and adhesion glycoproteins (Dammer et al, Science, 1995; 267:1173).
Separating biological molecules on the basis of their relative affinities for ligands is a well recognised technique. For example, in affinity chromatography, the components to be separated are passed down a column that contains a specific ligand. The component of interest binds preferentially and strongly to the column and is retained on the column while the other components are removed. The bound material may be eluted off the column at a later stage.
Separation technologies are an important part of many research experiments. Increasing the sensitivity or selectivity of these techniques is desirable.
Kolomenskii et al, J. Appl. Phys., 1998; 84(4):2404-10, discloses surface cleaning and adhesion studies conducted using laser-generated surface acoustic pulses. The pulses were at a low repetition rate (20 Hz) and constant energy. The procedure was conducted in vacuum, and therefore is not suitable for commercial exploitation. An optical microscope was used to detect the removal of particles and it was not possible to distinguish between particles of different size.
WO98/45692 discloses the use of a piezoelectric crystal sensor for determining the formation/dissociation of clathrate hydrates. Kurosawa et al, Chem. Pharm. Bull, 1990; 38(5):1117-20, reports using such a sensor for the detection of agglutination of antibody-bearing latex. WO98/40739 also discloses such a sensor, including a plate on which specific binding entities are immobilised, for use in indicating the presence of cells in a medium. These sensors are used by measuring a change in resonance frequency at constant voltage.
At present, where possible, most viruses are detected by culture of the specimen in cells, since this method is sensitive although time-consuming. Direct detection of viral DNA or RNA in clinical samples can be achieved using PCR and specific primers tailored for the virus of interest. Since PCR involves an amplification step, cross-contamination is a major problem and it is difficult to establish reliable quantitative methods. Other direct methods include electron microscopy, immune electron microscopy, and methods based on antigen detection with enzyme-linked antibodies. These methods are often relatively insensitive and hence require relatively large quantities of the viral particles.
Many biotechnological processes are based on specific properties, such as the binding affinities, of one or more biological or chemical entities. For example, separation techniques may aim to separate one or more different entities having specific properties from a sample. A biosensor or analytical method may aim to detect only chemical or biological entities having specific properties, which may be present in a sample.
In such processes, it is often important to discriminate between entities with similar properties. For example, a separation technique, such as affinity chromatography, or a biosensor, may need to discriminate between similar entities with only subtle differences therebetween. Examples of properties which can be used to discriminate between biological or chemical entities include their size, mass, isoelectric point, presence or absence of labels, composition, structure, or the presence of recognition sites to which specific binding means can bind.
Biotechnological processes can be made to be specific by including process steps that use binding means with specific affinity for biological or chemical entities. Example binding means include antibodies or antibody fragments, chemical ligands, or nucleic acid sequences which have affinity for chemical and biological entities including specific recognition sites to which binding means bind. For example, antibodies or antibody fragments bind to regions of their ligands referred to as epitopes.
Processes which are capable of discriminating between chemically similar entities on the basis of their aggregation are useful in fields including biochemistry, biotechnology, microbiology, polymer-science and materials science.
Chemical or biological entities which are similar to each other in many ways but which have different recognition sites can be discriminated between if binding means with different affinities for the different recognition sites can be found. However, it is not possible to discriminate between entities in this way if they have only similar or identical recognition sites, or if binding means able to discriminate with sufficient specificity between two similar entities cannot be found or are not commercially viable.
Thus, antibodies and other binding means for binding specific recognition sites often cannot discriminate between an entity that is present as an unaggregated single component and an entity that is present in the form of an aggregate of a plurality of components. This is because the component may have the same or similar binding properties (such as presenting the same or similar recognition sites to specific binding means) whether or not it is aggregated.
There is a great deal of interest in detecting diseases of the brain, in animals or humans in which proteins form aggregates within cells in afflicted individuals. For example, Alzheimer's Disease is characterised by the formation of aggregates of β-amyloid peptide. Such aggregates typically comprise proteins which have a normal, non-disease state form present in the cells of the central nervous system as discrete non-aggregated monomers, and also a disease-state form in which they can aggregate. The disease and non-disease state forms may differ only in terms of configuration, and/or may have chemical differences. It is therefore desirable to determine whether such proteins are present in aggregated or discrete non-aggregated (monomer) form. A discrete protein and an aggregate of many proteins will both have similar properties and the same, or similar, epitopes to which antibodies and other binding means can bind, and so are hard to discriminate between by virtue only of the affinity with which they are bound by antibodies.
Some diseases in which proteins form aggregates in the cells of sufferers are believed to be transmitted by proteinaceous infectious particles referred to as prions which are typically modified forms of mammalian proteins. It is desirable to detect these protein aggregates and to enable the protein aggregates to be discriminated from unaggregated (or less aggregated) prion proteins, whether in their non-disease state form or modified disease-state form. In some diseases, the infectious particle is thought to be an aggregate of proteins and it is desirable to detect this infectious aggregate.
In some applications, an entity which is separated from a sample will be detected or measured. Immunological techniques, such as sandwich immunoassays, are known which can quantify the number of recognition sites present in a sample. However, they cannot in general discriminate between whether these recognition sites are present within an aggregate or whether those recognition sites are present on individual components.
Aggregates comprising a plurality of components could in principle be discriminated from unaggregated or less aggregated components by virtue of their different masses. However, conventional mass-sensing methods used in biotechnology, such as mass spectrometry, surface plasmon resonance detection, the use of field effect transistors, or enzyme-linked immunosorbent assays (ELISAs), cannot discriminate between, for example, a) one aggregate comprised of one hundred thousand monomer sub-units, and b) one hundred thousand discrete monomer sub-units. In general, both a) and b) will have identical or similar masses, and will present similar, or identical, recognition sites to binding partners in similar or identical numbers.
A potential causative agent of Alzheimer's Disease is the 4-4.5 kDa, 39-43 residue β-amyloid (βA4) peptide. This peptide is proteoytically cleaved from three larger proteins encoded by alternative splicing of the β-amyloid protein precursor gene (βAPP). βAPP proteins are usually O- and N- linked glycosylated transmembrane proteins of 695, 751 and 770 residues with a 47 residue cytoplasmic domain. βA4 corresponds to 28 extracellular residues and 15-16 transmembrane residues of the βAPP proteins. Proteolytic processing of βAPP proteins at a position equivalent to residue 16 of βA4 usually leads to shedding of the extracellular domain. However, an inappropriate cleavage event leads to generation of soluble, cytoplasmic βA4. Fibrillar aggregates of βA4 are formed when the protein is transformed by partial denaturing to the beta-sheet configuration, but only very slowly at physiological pH of 7-7.5. The aggregation process is more rapid at the lower pH of 5-6 (as found in some sub-cellular compartments), but also requires additional factors such as radical generation ormetal-catalysed oxidation systems. It is the aggregation of βA4 into fibrillar bundles that ultimately leads to neuronal cell death and the onset of dementia.
Accordingly, it is desirable to detect βA4 aggregates, perhaps in the presence of either or both discrete unaggregated βA4 or correctly processed βAPP proteins.
A potential causative agent of Parkinson's Disease is α-synuclein, a 14 kDa protein which in non-disease-state form is an intrinsically unstructured/unfolded presynaptic protein. However, when it is oxidised at tyrosine or methionine residues, it enters a partially folded, disease-state form which accelerates its polymerisation to form amyloid-like fibrils. In Parkinson's Disease, these fibrils lead to degeneration of dopaminergic neurons of the substantia nigra and Parkinsonia motor deficits. There is genetic evidence for a direct role of alpha-synuclein in early onset, familial Parkinson's Disease, including mutations (G209A) that enhance its stability and propensity to cause fibrils. Accordingly it is desirable to detect the formation of aggregated alpha-synuclein (in polymerised form), perhaps in the presence of discrete alpha-synuclein proteins (whether unoxidised or oxidised, normal or mutant).
A potential causative agent of neurodegenerative Huntington's disease is the ubiquitously expressed 55-60 kDa huntington protein. The huntington protein has little homology to other proteins, but in the disease state is characterised by amplification of a CAG codon in the open reading frame, leading to glutamine repeats in the mature protein. This poly-glutamine region (comprising perhaps 80-100 glutamine repeats) in the full length protein leads to cytoplasmic aggregation, while smaller N-terminal poly-glutamine-rich fragments can form nuclear aggregations, resulting in neuronal death. It is therefore desirable to detect aggregates of the full-length protein or N-terminal fragments, perhaps in the presence of protein/protein fragments with fewer or no glutamine components.
In Creutzfeld-Jakob disease and Gerstmann-Straussler-Scheinker disease, the monomer prion protein exists in a ubiquitously-expressed, normal non-disease state cellular form (PrPc), and a refolded, protease resistant, heat resistant, infectious disease-state form. For example, in the disease-state form of ovine transmissible spongiform encephalopathy neurodegenerative disease (scrapie) is referred to a PrPSc. PrPSc is able to convert cellular PrPc to the infectious disease-state PrPSc form. It is believed that the protease resistance of PrPSc (and mutant form of PrPc, e.g. P105L, D178N-129N, T183A, F198S) leads to the sequentration of the protein in inclusion bodies, where it self-assembles into beta-sheet oligomers, ultimately forming fibrils characteristic of the neurodegenerative Creutzfeld-Jakob Disease, Gerstmann-Sträussler-Scheinker Syndrome and fatal familial insomnia.
It is therefore desirable to detect aggregates of disease-state forms of PrPc and other prion proteins, perhaps in the presence of unaggregated disease-state or non-disease-state forms of PrPc and other prion proteins.